Project Summary The overall objective of the U19 is to discover the role of the PD-1 pathway in human immunity by studying the effects of therapeutic PD-1 blockade on immune responses to chronic viruses (Project 1) and preventive vaccines (Project 2). To monitor adaptive immune responses in patients, we need to track the complex repertoire of T and B cells since clonotypes may behave differently over time and in response to infection and therapy. While there are several methods for tracking antigen-specific lymphocytes, large volumes of blood are needed for monitoring multiple antigens, biopsies have limited material for standard pipelines, and clonotypes with distinct antigen receptors are not distinguished. Standard bulk sequencing of TCRs and BCRs can provide quantitative clonotype frequencies; however, its utility is limited because the target antigens are not known for each TCR/BCR and the activation states of each clonotype cannot be determined. Core D provides an innovative service for tracking antigen-specific T and B cells in patient blood and tissues, allowing Projects 1 and 2 to quantify the frequency, antigen specificity and activation states of lymphocytes. Aim 1 provides an approach to match TCRs and BCRs to antigens by sequencing antigen receptors in patient T and B cells stimulated or tagged with viral or vaccine antigens. Aim 2 uses the more cost-effective bulk TCR- and BCR-seq to quantify the frequencies of clonotypes in serial samples of patients treated with anti-PD-1 therapy, but takes advantage of the results in Aim 1 to link antigen receptors from the repertoire with specific viral or vaccine antigens. Aim 3 uses leading-edge droplet-based RNA-seq to sequence paired antigen receptor chains (TCR? & ?; IgH & IgK/L) along with thousands of mRNAs in single lymphocytes, linking functional pathways with antigen receptors. The resulting dataset from the three Aims will be integrated to provide the frequencies, antigen specificities and activation states of T and B cells, allowing us to test the hypothesis that anti-PD-1 therapy differentially impacts the proliferation and activation of each lymphocyte subset depending on its antigen specificity and pre-therapy differentiation state. In short, Core D enables the monitoring of antigen- specific lymphocytes at unprecedented resolution, and will help dissect changes in immune protection against chronic infections (including HBV, HCV, CMV, and EBV), and influenza vaccination as result of anti-PD-1 therapy. The establishment and refinement of these approaches and creation of a Core D infrastructure will enable interrogation of immune mechanisms and provide insights into development and durability of immunological memory in the context of checkpoint blockade or any other immunotherapies.